Alternative splicing is a key mechanism for regulating genetic output that is directed by diverse pre-mRNA binding proteins. Recent genomic analyses have lent insight into the breadth of the regulatory networks controlled by these proteins. However, our mechanistic understanding of how the regulatory proteins affect the assembling spliceosome and their molecular interactions is still rudimentary. This information will be essential to the study of many human diseases attributed to misregulated splicing. In this project we examine how the polypyrimidine tract binding proteins, PTBP1 and PTBP2, and their cofactors control the splicing of neuron- specific exons. We will use stem cell differentiation protocols to examine two transitions in neuronal splicing regulation: one early in neuronal development when PTBP1 is replaced with PTBP2, and one occurring when PTBP2 is downregulated late in neuronal maturation, which was recently discovered. Using genomewide methods of RNA analysis, we will identify exons that are coregulated by combinations of PTBP1 or PTBP2 with different cofactors. From these datasets, sites of binding by PTBP1 or PTBP2 with particular cofactor proteins such as Matrin3, Raver1, and Mbnl1 will be identified and analyzed for assembly into regulatory RNPs in vitro. Genomewide screens will identify new cofactors affecting PTBP1 and/or PTBP2 activity, and we will compare exons controlled by PTBP1 and PTBP2 with the goal of understanding how the targeting of these two proteins differs. Non-canonical roles for PTB Proteins in regulating nuclear retention of unspliced transcripts and in neuronal microRNA processing will be explored. Finally, we will use biochemical methods to study the mechanisms by which the PTB proteins repress splicing. Building on systems developed in the last funding period, exon complexes assembled under different regulatory conditions will be purified to examine how the PTB proteins block spliceosome assembly. We will comprehensively identify interactions between components of these exon complexes, and characterize how specific regulators change these interactions. We will develop biochemical assays for the function of PTBP1/2 coregulators. These studies will yield new understanding of how RNA binding proteins work together to alter spliceosome assembly, and define new features of neuronal mRNA metabolism.